NME3 Human

What is NME3 and what are its primary functions in human cells?

NME3 (Nucleoside Diphosphate Kinase 3) is a protein encoded by the NME3 gene that serves dual critical functions in human cells. First, it functions as an NDP kinase, catalyzing the transfer of phosphate groups between nucleoside diphosphates and triphosphates. Second, it acts as a stimulator of mitochondrial fusion by interacting with mitofusins (MFN1/2) on the mitochondrial outer membrane .

The protein is localized to the mitochondrial outer membrane, dependent on its N-terminal region . This dual functionality allows NME3 to play crucial roles in both energy metabolism and mitochondrial dynamics, which are particularly important in cells with high energy demands such as neurons. The integration of these functions enables cells to adapt to metabolic stress conditions, particularly glucose starvation, where mitochondrial fusion and ATP production become essential for cell survival .

How does NME3 contribute to mitochondrial dynamics?

NME3 contributes to mitochondrial dynamics primarily by promoting mitochondrial fusion. Located on the mitochondrial outer membrane, NME3 interacts directly with mitofusins (MFN1/2), which are GTPases essential for the fusion of mitochondrial outer membranes . This interaction facilitates the fusion process, leading to elongated mitochondrial networks.

Research using patient-derived fibroblasts with NME3 deficiency has demonstrated that these cells exhibit a significantly slower rate of mitochondrial dynamics compared to normal cells . This dysfunction can be reversed by expressing either wild-type or catalytic-dead NME3, suggesting that NME3's role in promoting fusion is independent of its NDP kinase activity . Further evidence comes from knockdown experiments in normal human fibroblasts and HeLa cells, which resulted in reduced rates of mitochondrial fusion, confirming NME3's direct involvement in this process .

What is known about NME3 expression patterns in human tissues?

While the search results don't provide comprehensive information about NME3 expression patterns across all human tissues, they do indicate that NME3 plays a particularly critical role in neuronal development and function. This is evidenced by the fatal neurodegenerative disorder associated with NME3 deficiency, which presents with congenital hypotonia, hypoventilation, and cerebellar histopathological alterations .

The sensitivity of neuronal tissue to NME3 deficiency suggests that neurons may have higher expression levels or greater dependency on NME3 function compared to other cell types. This aligns with the understanding that neurons have high energy demands and rely heavily on proper mitochondrial function and dynamics for survival . Additionally, the regulation of mitochondrial morphology through fusion and fission processes is particularly important for neuronal development and function, explaining why NME3 deficiency manifests primarily as a neurodegenerative disorder .

Further research on tissue-specific expression patterns of NME3 would provide valuable insights into its differential roles across human tissues and potentially explain the tissue-specific pathology observed in NME3-related disorders.

How do the dual functions of NME3 cooperate during metabolic stress?

NME3 exhibits a remarkable functional duality that becomes particularly significant during metabolic stress conditions such as glucose starvation. The two separate functions—stimulation of mitochondrial fusion and NDP kinase activity—cooperate in a coordinated manner to ensure cell survival .

During glucose starvation, wild-type cells typically respond by increasing mitochondrial fusion, which enhances the efficiency of the respiratory electron transfer chain. In this process, NME3 plays a critical role by interacting with MFN1/2 to promote mitochondrial elongation, creating a more interconnected mitochondrial network . This structural adaptation is the first line of defense against energetic stress.

Simultaneously, NME3's NDP kinase activity becomes crucial for maintaining adequate levels of nucleoside triphosphates, particularly GTP, which is essential for numerous cellular processes including protein synthesis and signal transduction . The research conducted on patient-derived cells has shown that while both wild-type and catalytic-dead NME3 can restore mitochondrial elongation under glucose starvation, only wild-type NME3 (with intact NDP kinase activity) could sustain ATP production and cellular viability .

This indicates a sequential mechanism where NME3 first promotes structural adaptation through mitochondrial fusion, and then supports metabolic adaptation through its enzymatic activity. The integration of these functions allows for a comprehensive cellular response to metabolic stress, highlighting why NME3 deficiency leads to catastrophic consequences in high-energy-demanding tissues like the brain .

What is the relationship between NME3 mutations and neurodegenerative disorders?

The relationship between NME3 mutations and neurodegenerative disorders has been established through the identification of a homozygous mutation in the initiation codon of the NME3 gene in patients with a fatal neurodegenerative condition . This mutation leads to deficiency in NME3 protein expression and manifests as congenital hypotonia, hypoventilation, and cerebellar histopathological alterations .

The neurodegenerative phenotype can be explained by several interconnected factors. First, the central nervous system has exceptionally high energy demands, making it particularly vulnerable to disruptions in energy metabolism caused by NME3 deficiency . Second, proper mitochondrial dynamics are crucial for neuronal development and function, including axonal transport, synaptic transmission, and calcium buffering .

At the cellular level, patient-derived fibroblasts exhibited abnormal mitochondrial morphology with altered cristae structure and dysfunction in mitochondrial dynamics . These cells were also highly sensitive to glucose starvation, showing rapid mitochondrial fragmentation and cell death instead of the adaptive fusion response seen in normal cells .

The specificity of the neurological phenotype suggests that while NME3 may have broader functions, neurons are either more dependent on its activity or less able to compensate for its loss through alternative pathways . The progressive nature of the disorder further indicates that cumulative mitochondrial dysfunction, potentially exacerbated by metabolic stress events, leads to neuronal death and degeneration .

This link between NME3 and neurodegeneration provides important insights into the critical role of mitochondrial dynamics and energy metabolism in neuronal health and offers potential therapeutic targets for related disorders.

How does NME3 interact with the DRP1-dependent mitophagy pathway under hypoxic conditions?

Recent research has identified NME3 as a gatekeeper for DRP1-dependent mitophagy under hypoxic conditions . While the search results don't provide detailed information about this specific interaction, we can infer some key aspects based on our understanding of mitochondrial dynamics and the established functions of NME3.

DRP1 (Dynamin-related protein 1) is a key mediator of mitochondrial fission, which is often a prerequisite for mitophagy—the selective degradation of damaged or dysfunctional mitochondria. Under hypoxic conditions, cells typically need to adjust their mitochondrial content and function to adapt to reduced oxygen availability .

Based on NME3's established role in promoting mitochondrial fusion by interacting with MFN1/2 , it likely acts as a counterbalance to DRP1-mediated fission. As a "gatekeeper" for DRP1-dependent mitophagy, NME3 may regulate when and to what extent mitochondria undergo fission and subsequent mitophagy under hypoxic stress .

This regulatory role would be consistent with NME3's broader function in metabolic adaptation, where it helps determine whether mitochondria should be preserved through fusion (promoting energy efficiency) or eliminated through fission and mitophagy (removing damaged organelles) .

The identification of NME3 as a regulator of hypoxia-induced mitophagy opens new avenues for understanding how cells balance mitochondrial dynamics in response to oxygen limitation, with potential implications for conditions involving tissue hypoxia, such as ischemic diseases and cancer .

What experimental models are most appropriate for studying NME3 function?

Selecting appropriate experimental models for studying NME3 function requires consideration of the specific research questions and the biological contexts in which NME3 operates. Based on the available research, several effective models can be identified:

Patient-derived fibroblasts: The studies described in the search results successfully utilized fibroblasts from patients with NME3 mutations . These cells provide a physiologically relevant model with complete NME3 deficiency, allowing researchers to study the consequences of NME3 loss in human cells. They're particularly valuable for investigating mitochondrial dynamics, cellular responses to metabolic stress, and rescue experiments with various NME3 constructs .

Knockdown approaches in established cell lines: Both normal human fibroblasts and HeLa cells with NME3 knockdown have been used to validate findings from patient-derived cells . These models offer the advantage of controlled NME3 reduction in well-characterized cell types, facilitating mechanistic studies.

Reconstitution experiments: Expression of wild-type, catalytic-dead, or oligomerization-attenuated NME3 in deficient cells has proven instrumental in dissecting the separate functions of NME3 . These experiments allow researchers to determine which protein domains and functions are required for specific cellular processes.

Metabolic stress paradigms: Glucose starvation protocols have been particularly informative in revealing NME3's role in metabolic adaptation . Similarly, hypoxia models can be employed to study NME3's function in DRP1-dependent mitophagy .

Mitochondrial imaging techniques: Given NME3's role in mitochondrial dynamics, fluorescent labeling of mitochondria combined with live-cell imaging is essential for monitoring fusion and fission events, mitochondrial morphology, and dynamics rates .

For more comprehensive in vivo studies, animal models with conditional NME3 knockout, particularly in neuronal tissues, would be valuable but were not specifically mentioned in the search results.

What techniques are essential for analyzing mitochondrial dynamics in the context of NME3 research?

Analyzing mitochondrial dynamics in the context of NME3 research requires a combination of advanced imaging, biochemical, and molecular techniques:

Live-cell imaging: This is fundamental for directly observing mitochondrial fusion and fission events in real-time. Fluorescent markers targeting mitochondria (such as MitoTracker dyes or mitochondrially-targeted fluorescent proteins) can be used to visualize morphological changes and calculate rates of mitochondrial dynamics . Time-lapse microscopy allows for tracking these events over time in response to various conditions or treatments.

Electron microscopy: As employed in the studies of patient fibroblasts, electron microscopy provides ultra-structural details of mitochondrial morphology, including cristae organization, which was found to be abnormal in NME3-deficient cells . This technique offers resolution beyond the capabilities of light microscopy.

Photoactivatable fluorophores: Techniques using photoactivatable GFP targeted to mitochondria can enable the tracking of specific mitochondrial populations after photoactivation, allowing precise measurement of fusion rates.

Co-immunoprecipitation and protein interaction assays: These techniques have been crucial in establishing NME3's interaction with MFN1/2 on the mitochondrial outer membrane . They remain essential for identifying additional interaction partners in the mitochondrial dynamics machinery.

Mitochondrial fractionation: Subcellular fractionation to isolate mitochondria, followed by proteomic analysis, can help determine the precise localization of NME3 within mitochondrial compartments and identify associated proteins .

Functional assays under stress conditions: Applying metabolic stressors such as glucose starvation has been particularly informative in revealing NME3's role in adaptive responses . Measuring parameters such as ATP production, oxygen consumption, and cell viability under these conditions provides functional readouts of mitochondrial performance.

Gene expression and protein analysis: Quantifying the expression levels of mitochondrial dynamics proteins (MFN1/2, DRP1, OPA1) in response to NME3 manipulation can reveal regulatory relationships and compensatory mechanisms .

By combining these techniques, researchers can comprehensively analyze how NME3 influences mitochondrial dynamics at structural, functional, and regulatory levels.

How can researchers effectively study the dual functions of NME3 in mitochondrial fusion and as an NDP kinase?

Studying the dual functions of NME3 effectively requires strategic experimental design that can separate and evaluate each function independently while also assessing their cooperative effects. Based on the research approaches described in the search results, several methods have proven valuable:

Domain-specific mutant constructs: The use of catalytic-dead NME3 (which lacks NDP kinase activity but retains the ability to promote fusion) and oligomerization-attenuated NME3 (which affects its structural role in fusion) has been instrumental in dissecting these separate functions . Creating a panel of such domain-specific mutants allows researchers to attribute specific cellular outcomes to distinct protein functions.

Function-specific rescue experiments: Introducing these various NME3 constructs into NME3-deficient cells (such as patient-derived fibroblasts) and measuring different outcomes provides clear evidence of function-specific effects . For example, the research showed that both wild-type and catalytic-dead NME3 restored mitochondrial elongation, but only wild-type NME3 sustained ATP production and viability .

Metabolic stress paradigms: Glucose starvation has been particularly effective in revealing the cooperative nature of NME3's dual functions, as this stress requires both mitochondrial fusion and efficient energy production for cell survival . Similar stress paradigms that challenge both mitochondrial dynamics and energy metabolism can help elucidate how these functions interact.

Biochemical activity assays: Direct measurement of NDP kinase activity using purified proteins or immunoprecipitated complexes can quantify the enzymatic function independently of morphological effects on mitochondria .

Live-cell imaging combined with metabolic measurements: Simultaneous monitoring of mitochondrial morphology (through fluorescent imaging) and metabolic parameters (such as ATP levels, oxygen consumption, or NADH/NAD+ ratios) can reveal how structural changes correlate with bioenergetic outcomes .

Proximity labeling techniques: Methods such as BioID or APEX2 could identify proteins that interact with NME3 in different functional contexts, potentially revealing separate protein complexes associated with its fusion-promoting versus enzymatic activities .

Temporal analysis of responses: Determining the sequence of events following NME3 activation or in response to stress can reveal whether one function precedes and potentially enables the other, providing insight into their mechanistic relationship .

By employing these complementary approaches, researchers can deconvolute NME3's dual functions and understand how they integrate to support cellular adaptation and survival.

What therapeutic strategies might target NME3 pathways in neurodegenerative disorders?

Given the critical role of NME3 in mitochondrial dynamics and energy metabolism, particularly in neuronal tissue, several potential therapeutic strategies emerge that could target NME3-related pathways in neurodegenerative disorders:

Gene therapy approaches: For patients with homozygous mutations in the NME3 gene, targeted gene replacement or supplementation could restore functional NME3 protein expression . This approach would address the root cause of the disorder, particularly if delivered early in development before irreversible neuronal damage occurs.

Mitochondrial fusion promoters: Since NME3 deficiency leads to impaired mitochondrial fusion, compounds that directly promote mitochondrial fusion through MFN1/2 activation could potentially bypass the need for NME3 . Recent research has identified several small molecules capable of promoting mitochondrial fusion that could be investigated in NME3-deficient models.

Metabolic modulators: Given that NME3-deficient cells are particularly vulnerable to glucose starvation, interventions that enhance alternative energy production pathways might provide protection . This could include ketogenic diets or compounds that increase mitochondrial efficiency through alternative mechanisms.

NDP kinase activity enhancers: For mutations that specifically impair the enzymatic function of NME3 while preserving its structural role in fusion, compounds that enhance the remaining NDP kinase activity or supplement GTP levels might ameliorate some aspects of cellular dysfunction .

Mitophagy regulators: Based on NME3's newly identified role as a gatekeeper for DRP1-dependent mitophagy under hypoxic conditions , compounds that modulate mitophagy could potentially compensate for NME3 dysfunction. This could involve either enhancing the selective removal of damaged mitochondria or preventing excessive mitophagy, depending on the specific pathology.

Cell-based therapies: For severe neurodegenerative conditions associated with NME3 mutations, transplantation of healthy neuronal precursors or supportive cells (like astrocytes) with normal NME3 function might provide trophic support and functional improvement .

Any therapeutic approach would need to consider the developmental timing of intervention, as the congenital nature of the reported NME3-related disorder suggests that early intervention would be crucial for preventing irreversible neurological damage .

How might NME3 function differ across various cell types and physiological conditions?

The differential effects of NME3 deficiency across tissues, with particularly severe consequences in the nervous system, suggest that NME3 function may vary significantly across cell types and under different physiological conditions . Several factors likely contribute to this variation:

Metabolic demands: Neurons have exceptionally high energy requirements and limited glycolytic capacity, making them particularly dependent on efficient mitochondrial function and thus potentially more sensitive to NME3 deficiency . Other cell types with high energy demands, such as cardiomyocytes or skeletal muscle cells, might similarly rely heavily on NME3 function, though this wasn't specifically addressed in the search results.

Developmental stage-specific requirements: The congenital presentation of NME3-related neurological disorders suggests a critical role during development . NME3's importance may vary throughout the lifespan, with potentially greater significance during periods of high metabolic demand or stress, such as development, growth, or aging.

Stress adaptation capacity: Under glucose starvation, NME3 becomes critical for adapting mitochondrial dynamics and maintaining energy production . Similarly, its role in DRP1-dependent mitophagy under hypoxia suggests differential importance under stress conditions . Other physiological stressors like oxidative stress, temperature fluctuations, or inflammation might similarly modulate NME3 dependency.

Compensatory mechanisms: Some cell types may have redundant systems that can partially compensate for NME3 deficiency, such as other NME family members or alternative pathways for regulating mitochondrial dynamics . The availability and effectiveness of these compensatory mechanisms likely vary across tissues.

Mitochondrial network baseline characteristics: Cell types naturally differ in their baseline mitochondrial morphology and dynamics rates. Those with more dynamic networks or greater reliance on fusion-fission balance for function might be more affected by NME3 dysfunction .

Interaction with tissue-specific factors: NME3 may interact with tissue-specific proteins or be subject to tissue-specific regulatory mechanisms that modify its function or importance in different cellular contexts .

Further research specifically comparing NME3 function and requirements across different cell types, developmental stages, and stress conditions would provide valuable insights into its context-dependent roles and the selective vulnerability of certain tissues to its dysfunction.

What are the potential connections between NME3 and other mitochondrial dynamics proteins in human disease?

NME3's interactions with the mitochondrial dynamics machinery suggest potential connections with other proteins involved in these processes, with implications for various human diseases:

Mitofusins (MFN1/2): NME3 directly interacts with MFN1/2 to promote mitochondrial fusion . Mutations in MFN2 cause Charcot-Marie-Tooth disease type 2A, a peripheral neuropathy. Given their functional interaction, NME3 could potentially modify the severity or presentation of MFN2-related disorders, and vice versa. Patients with partial deficiencies in both proteins might exhibit synergistic pathology.

DRP1 and mitophagy pathways: NME3's newly identified role as a gatekeeper for DRP1-dependent mitophagy under hypoxia suggests functional interactions with the mitochondrial fission and mitophagy machinery . Dysregulation of DRP1 has been implicated in various neurodegenerative conditions including Alzheimer's and Huntington's diseases. NME3 dysfunction might exacerbate pathology in these conditions by disrupting the balance of mitochondrial dynamics.

OPA1 and cristae structure: The abnormal cristae morphology observed in NME3-deficient cells parallels defects seen in OPA1 dysfunction, which causes dominant optic atrophy . Although not directly addressed in the search results, NME3 might indirectly influence OPA1 function or cooperate in maintaining proper cristae organization. The related NME family member NME4 has been shown to associate with OPA1 .

PINK1/Parkin pathway: This pathway regulates mitophagy and is mutated in certain forms of Parkinson's disease. Given NME3's involvement in mitophagy regulation under hypoxia , there could be functional interactions or compensatory relationships between NME3 and the PINK1/Parkin system in mitochondrial quality control.

Metabolic sensors and regulators: NME3's dual role in both structure and metabolism suggests potential interactions with metabolic sensors like AMPK or mTOR, which are implicated in various metabolic diseases and cancer . These interactions could provide mechanistic links between mitochondrial dynamics and broader cellular metabolic regulation.

Other NME family members: Functional redundancy or compensation between NME3 and other NME family proteins might influence disease presentation and progression . Understanding these relationships could help explain variable penetrance or expressivity in NME-related disorders.

Exploring these potential connections could reveal new disease mechanisms and identify opportunities for therapeutic intervention across multiple disorders involving mitochondrial dysfunction. It might also explain why certain genetic variants or environmental factors modify the risk or progression of mitochondrial-related diseases.

What are the key challenges in measuring NME3 enzymatic activity in different cellular compartments?

Accurately measuring NME3 enzymatic activity in different cellular compartments presents several technical challenges that researchers must address:

Subcellular fractionation purity: NME3 is primarily localized to the mitochondrial outer membrane, but contamination during isolation of mitochondrial fractions can lead to inaccurate activity measurements . Developing highly pure fractionation methods that preserve NME3's native interactions and activity is essential.

Maintaining native protein complexes: NME3's NDP kinase activity may be influenced by its interactions with other proteins, such as MFN1/2 . Traditional protein purification methods might disrupt these interactions, leading to measurements that don't reflect the true in vivo activity. Gentle isolation techniques that preserve protein complexes are needed.

Compartment-specific nucleotide pools: NME3 transfers phosphate between different nucleoside diphosphates and triphosphates, but the availability and ratios of these substrates likely vary between cellular compartments . Accurately replicating these compartment-specific conditions in vitro is challenging but necessary for physiologically relevant activity measurements.

Temporal dynamics: NME3 activity may fluctuate rapidly in response to metabolic changes or stress conditions . Developing methods that can capture these temporal dynamics, rather than just steady-state activity, would provide more comprehensive insights into NME3 function.

Distinguishing from other NDP kinases: Cells express multiple NDP kinases with similar catalytic activities, making it difficult to specifically measure NME3 contribution in complex samples . Developing highly specific activity assays, possibly using NME3-specific antibodies or inhibitors, is necessary to overcome this challenge.

In situ activity measurements: Ideally, measuring NME3 activity without disrupting cellular architecture would provide the most accurate results. Developing fluorescent sensors or other non-invasive methods to monitor NDP kinase activity in living cells and specific compartments represents an important technical frontier.

Correlation with physiological outcomes: Connecting measured enzymatic activity with functional outcomes such as ATP production, GTP availability, or mitochondrial fusion rates is essential for understanding the biological significance of variations in NME3 activity . Integrated approaches that simultaneously measure enzyme activity and its downstream effects are needed.

Addressing these challenges will require innovative experimental approaches and may necessitate the development of new tools specifically designed for studying compartmentalized enzymatic activities in intact cellular systems.

How can researchers distinguish between the effects of NME3 on mitochondrial structure versus its enzymatic activity?

Distinguishing between NME3's effects on mitochondrial structure versus its enzymatic activity requires experimental strategies that can selectively manipulate each function. Based on the approaches described in the search results, several effective methods can be implemented:

Mutant construct analysis: The use of domain-specific NME3 mutants has been particularly valuable . Catalytic-dead NME3 (retaining structural function but lacking enzymatic activity) and oligomerization-attenuated NME3 (affecting structural function) allow researchers to separate these functions experimentally . By expressing these constructs in NME3-deficient cells and measuring specific outcomes, researchers can attribute effects to either function.

Temporal analysis of responses: Determining the timeline of events following NME3 activation or inhibition can help distinguish primary from secondary effects. If structural changes consistently precede metabolic alterations (or vice versa), this temporal relationship provides insight into causal connections between NME3's dual functions .

Chemical-genetic approaches: Developing small molecules that selectively inhibit NME3's enzymatic activity without affecting its protein-protein interactions (or vice versa) would provide valuable tools for acute, selective manipulation of each function in intact cells.

Correlative microscopy and functional assays: Combining high-resolution imaging of mitochondrial morphology with simultaneous measurements of local nucleotide concentrations or NDP kinase activity could reveal spatial and temporal correlations between structural and enzymatic functions .

Specificity controls: Comparing NME3 effects with those of other mitochondrial fusion promoters (that lack NDP kinase activity) or other NDP kinases (that don't promote fusion) can help distinguish function-specific outcomes .

Conditional manipulation systems: Developing systems that allow rapid, inducible control of NME3 enzymatic activity (e.g., through chemical inhibition or light-activated domains) would enable researchers to observe immediate consequences of activity changes before compensatory structural alterations occur.

Isolation of reaction products: Measuring the specific nucleotide triphosphates generated by NME3's kinase activity and correlating their levels with particular cellular processes can help attribute certain outcomes specifically to the enzymatic function .

Mathematical modeling: Developing computational models that incorporate both the structural and enzymatic functions of NME3 can help predict and distinguish their relative contributions to observed phenotypes under various conditions.

By implementing these complementary approaches, researchers can systematically dissect the distinct contributions of NME3's structural and enzymatic functions while also understanding how they integrate in physiological contexts.

What control experiments are crucial when studying NME3 in patient-derived cells?

When studying NME3 in patient-derived cells, several critical control experiments must be included to ensure reliable and interpretable results:

Genetic confirmation: Verification of the specific NME3 mutation through sequencing in patient-derived cells is essential, along with confirmation that this mutation indeed results in NME3 protein deficiency through Western blotting or immunofluorescence . This establishes the fundamental premise of the experimental system.

Healthy control cells: Including cells from healthy individuals with similar genetic backgrounds (ideally unaffected family members when possible) provides the most appropriate control for comparison . Age-matched controls are particularly important when studying conditions that may have developmental components.

Rescue experiments: Reintroducing wild-type NME3 into patient-derived cells should reverse the observed phenotypes if they are truly caused by NME3 deficiency . This is perhaps the most critical control experiment as it directly establishes causality between NME3 loss and observed abnormalities.

Domain-specific mutant controls: Including catalytic-dead or oligomerization-attenuated NME3 variants in rescue experiments helps distinguish which specific functions of NME3 are necessary for particular cellular processes . This provides mechanistic insight beyond simple rescue.

NME3 knockdown in control cells: Reducing NME3 expression in healthy control cells through siRNA or CRISPR techniques should recapitulate key phenotypes observed in patient cells, further confirming the specificity of NME3's role .

Cell type-matched controls: Given potential variability in NME3 function across cell types, using the same cell type (e.g., skin fibroblasts) for both patient and control samples ensures appropriate comparison .

Passage number matching: Controlling for cell passage number is important, as extended culture can affect mitochondrial function and cellular phenotypes independently of genetic differences.

Environmental stress controls: When studying responses to stressors like glucose starvation, it's crucial to verify that control cells show the expected adaptive responses (e.g., mitochondrial elongation) under identical conditions . This confirms that the experimental conditions are appropriate for observing the phenotypes of interest.

Mitochondrial function baseline measurements: Establishing baseline measurements of mitochondrial membrane potential, respiratory capacity, and ATP production helps distinguish primary effects of NME3 deficiency from secondary consequences .

Independent measurement techniques: Utilizing multiple independent techniques to measure the same parameters (e.g., assessing mitochondrial morphology by both fluorescence microscopy and electron microscopy) increases confidence in the findings .